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K. YOSHINAGA

Summary.

The effect of progesterone or oestradiol on the delay in implantation in lactating rats was studied with a new technique for local application. Both progesterone and oestradiol abolish the delay. A localized implantation is induced at the normal time by local application of progesterone at around 5 μg, but further increase of the dose up to 100 μg did not increase the area of the uterus affected by the hormone. On the other hand, in the application of oestradiol, the larger the dose, the larger the region of uterus affected by the hormone, resulting in various degrees of responses from local to systemic positive with increase of dose from 0·002 to 0·1 μg.

The cause of the delay in implantation during lactation and possible mechanisms are discussed.

K. YOSHINAGA and C. E. ADAMS

Psychoyos (1961) examined the development of eggs which had been collected from donor rats, that were either normally pregnant or undergoing delayed implantation, and then transferred to the uteri of spayed and progesterone treated recipients. It was found that implantation of such eggs occurred only after the recipient had been treated with oestrogen. Later, he established the importance of the timing of oestrogen treatment in relation to egg transfer (Psychoyos, 1963). We have now examined the fate of blastocysts transferred to the uteri of rats, which were treated in several different ways, so as to obtain further information on the hormonal conditions affecting implantation.

A total of sixty-five adult hooded rats was used as recipients. Eggs were obtained from donors during the morning of Day 5 of pregnancy (Day 1 = day on which spermatozoa

K. YOSHINAGA and C. E. ADAMS

Summary.

Blastocysts were transferred reciprocally between the rabbit and rat. Rat blastocysts either with or without the zona pellucida survived transfer to the oviduct of the pseudopregnant rabbit for 48 hr, as demonstrated by their capacity to implant upon re-transfer to rats; however, only a few survived to term. Rabbit blastocysts degenerated when transferred for 48 hr to the uterus of rats in the 'delayed implantation' condition.

Whilst the tumour grew well outside the uterus in all groups, tumour tissue only invaded the uterine wall in Groups 5 and 6. Growth was most marked in the pseudopregnant animals, where the tumour was actively invading the anti-mesometrial decidua. In Groups 3 and 5, the uterine lumen was full of extravasated blood and polymorphonuclear leucocytes, whereas in Groups 1, 2 and 4 the uterus appeared normal, with no signs of any tissue reaction.

It is concluded that the tumour behaves very like the blastocyst; its ability to survive within the uterus is hormone dependent, whereas it can develop outside the uterus irrespective of the hormonal environment. The mechanisms by which a hostile endometrium can destroy the tumour are not known.

K. YOSHINAGA and C. E. ADAMS

The influence of the time of spaying on the occurrence of delayed implantation has been intensively studied in the rat. Although it was originally reported that early spaying (Day 1 or 2 of pregnancy) followed by progesterone treatment did not lead to a delay in implantation, whereas spaying at a later stage (Day 4) did (Canivenc, Laffargue & Mayer, 1956; Cochrane & Meyer, 1957), further work on rats revealed that spaying before Day 4 generally resulted in delayed implantation (Psychoyos & Alloiteau, 1962; Nutting & Meyer, 1963; Mayer, 1963). Failure to observe delayed implantation more universally in the early experiments has been ascribed in part to incomplete ovariectomy. As the influence of the time of spaying on implantation has apparently not been examined in the adult mouse, the present work was undertaken. Endocrinolo

K. YOSHINAGA and C. E. ADAMS

Summary.

Luteotrophic activity of the young conceptus was investigated on the basis of the maintenance of pregnancy produced artificially by means of egg transfer to the uteri of adult `cycling' rats treated with progesterone for 5 to 11 days. Pregnancy rate varied according to the duration of progesterone treatment; the longer the treatment, the higher the rate of success. Luteotrophic activity of the conceptus appeared soon after implantation and increased gradually to the time of establishment of the foetal placenta.

K. YOSHINAGA, W. A. MAHONEY and G. PINCUS

Hamner & Fox (1969) have recently reviewed various techniques for the collection of oviduct fluid in the rabbit, sheep, pig and monkey. Continual collection of the oviduct fluid from the rhesus monkey was first achieved by Mastroianni, Shah & Abdul-Karim (1961). They cannulated the oviducts with silicone rubber tubings which were connected to an external volumetric collection device fixed to the skin. Movements of the animals' upper limbs were restricted with plaster to prevent the animals from reaching the device. Using this technique, daily samples were collected for as long as four complete menstrual cycles.

In view of the possibility that a physical restriction of the monkey might cause a deleterious effect on the normal secretory patterns of the fluid, we developed a device which allows daily collection of the fluid from unrestricted monkeys. The device was installed intra-abdominally and samples were taken for a period of over 3 months, after which, however, the device was subject to rejection by the animal. The technique and preliminary data obtained are described in this report.

D. K. Saxena, I. Tanii, K. Yoshinaga and K. Toshimori

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml−1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.

K Yoshinaga, DK Saxena, T Oh-oka, I Tanii and K Toshimori

The monoclonal antibody mMN9 recognizes an antigenic molecule, equatorin, which is localized at the equatorial segment of the mammalian sperm acrosome. Our previous results using an IVF system indicated that mMN9 blocked sperm-oocyte fusion. Antibody-containing and control solutions were injected directly into the right and left oviductal ampullae, respectively, of anaesthetized female mice to assess the effect of mMN9 on fertilization in vivo. After hCG treatment, the females were mated, and their oviductal eggs and implanted embryos were examined. mMN9 was retained in the oviductal lumen at 20 h after injection. The rates of fertilization and concomitant pregnancy were significantly lower than in the control side (P < 0.05). In addition, histological studies showed no evidence of pathological changes in the female reproductive tract after the injections. These results indicate that mMN9 inhibits mouse fertilization significantly under in vivo conditions and that this injection method should be useful for studying the effects of antibodies and agents on fertilization in vivo.